Project Summary. Despite strong interest, the study of the 3D structures of biomolecules and their dynamics remain challenging by the inherent difficulty in growing 3D crystals suitable for X-ray diffraction and by their poor solubility for solution NMR studies. We propose a transient 2D IR approach that will address questions of conformational dynamics and structural change of backbone and side chain motions directly, especially when the biomolecule begins in a well-defined initial condition, and then upon short pulse photolysis, evolution of the resulting structure distributions can be tracked by 2D IR spectroscopy. In the course of this research, a spectroscopic tool will be developed to map out both structural motions while concurrently providing insight into the solvent dynamics at each labelled site and how their corresponding locations promote the molecular recognition and self-assembly through weak associative forces. The fast dynamics during the key structural events in RNA or antimicrobial peptide (AMP) action will be measured on time scales ranging from single bond rotational periods (fs-ps) to those required for significant conformational reorganization (ns-ms) by employing our transient 2D IR methods. Observations in real time of the non-equilibirum dynamics will provide an atomic level view of how chosen structures traverse reaction paths to stable final states. This information will then be used to challenge and test cutting edge non-equilibrium molecular dynamics simulations. The research outlined herein aims to combine techniques (eg. photo-initation, pH-jump, etc.) traditionally used to determine kinetics in linear spectroscopies with the information package that comes from probing with 2D IR spectroscopy. 2D IR spectroscopy will afford sufficient structural and time resolution to generate snapshots of molecular motions along the reaction pathway of specific biological events. In particular, we will simultaneously measure distances and angles within biomolecules and also detect the local vibrational dynamics, including H-bond exchange, coupled water dynamics and polar residue field fluctuations, around each individual probe. By harnessing the strengths of various initiation techniques, we will dissect the side chain motions and global structural changes responsible for molecular recognition, folding, and molecular assembly of AMP activity. Furthermore, we will disentangle the loss of hydrogen bonding, base stacking, and evolving compactness to uncover molecular details of the mechanistic pathway of RNA folding/unfolding. The broader objective is to obtain a chemical bond scale description of interactions that lead to productive conformational changes. Although RNA misfolds are believed to be responsible for autoimmune diseases such as lupus, they are not as well understood as protein misfolds leading to Alzheimer's disease for example. This work will help uncover the reasons for these non-native folds. Moreover, in regards to AMPs, some of these lytic peptides may hold the key to destroy cancer cells and mark the way for the development of therapeutics that can target specific lipid composition. Administrative Equipment Supplement Justification. As mentioned above, the parent proposal concerns quasi- or non-equilibrium dynamics of the interactions of peptides with various membrane mimics and RNA folding/unfolding measured via 2D IR and transient 2D IR nonlinear laser spectroscopy. To perform these experiments, a Coherent Libra ultrafast Ti:Sapphire amplifier (with 80fs pulses) is utilized to ultimately generate the mid-IR pulses necessary for the 2D IR photon echo measurements. This equipment supplement is requested for purchasing a replacement Evolution 30 laser diode head. The Evolution 30 laser diode head is the major component of the pump laser required for stable amplification of the Coherent Libra femtosecond laser system. Without efficient amplification, it is impossible to generate the stable femtosecond pulses necessary for all 2D IR and transient 2D IR measurements relevant to the parent grant (R15GM1224597). After 5 years, the laser diodes start to fail resulting in a reduction of conversion efficiency eventually leading to the inability to pump the amplifier system appropriately, ultimately creating instabilities. The original laser system was purchased with the PI's university startup funds and it was exactly 5 years ago. Thus, due to some recent drops in overall efficiency and a discussion with the Coherent laser technician, it was agreed that the laser diode heads are close to the end of their lifetime and a new assembly with installation costing $46,804 was eminent. So, it is with this justification that I am requesting the aforementioned equipment supplement.